Estrogen (E) deficiency is the major cause of postmenopausal osteoporosis. Thus, understanding the mechanisms by which E regulates bone metabolism is critical for developing novel approaches to prevent and treat this disorder. This project focuses on better defining key, unresolved issues regarding E action on bone in women. While previous studies have demonstrated that E deficiency is associated with increased RANKL expression by osteoblastic cells, a major unresolved issue regarding the increase in bone resorption in women following the menopause is whether increased resorption is solely due to E deficiency or is augmented by the concomitant rise in circulating follicle-stimulating hormone (FSH) levels. Thus, in Specific Aim 1 we will test the hypothesis that FSH directly regulates bone resorption independently of E. In addition to regulating bone resorption, it is clear that E also has important effects on maintaining bone formation. In Specific Aims 2 and 3, we focus on defining mechanisms for the age-related decrease in bone formation and the role of E deficiency in mediating this decrease. We will couple two novel in vivo human experimental paradigms with methods we have established to examine gene expression in highly purified bone marrow osteoblastic cells. Using quantitative polymerase chain reaction assays complemented by assessment of key functional and protein measurements by flow cytometry, we will test the hypothesis that there are both Edependent and E-independent defects in bone formation in aging women. In Specific Aim 2, we will test whether the decrease in bone formation we have previously observed following acute E deficiency in women is associated with a decrease in markers of Wnt/BMP signaling and/or production and in other genes related to bone formation by osteoblastic cells. In Specific Aim 3, we will test the hypothesis that there is an Eindependent defect in bone formation in elderly women and identify potential novel mechanisms for this agerelated impairment in bone formation. Collectively, these studies will lead to a better understanding of E action on bone, and are highly clinically relevant since they may identify new therapeutic targets to prevent and treat osteoporosis.